Project Summary Membranes provide a protective barrier to life, compartmentalize environments to promote desired reactions, and sift through their surroundings to sort molecules in and out of their compartments. Proteins have evolved to restructure membranes for different purposes such as vesicle trafficking, cell division, and organelle architecture. Organisms have also evolved proteins that disrupt membranes, serving as defense mechanisms against competitors and pathogens. We are beginning to understand how these proteins function at a structural and cell biological level in part due to advancements in electron microscopy. To gain more insight into how proteins disrupt membrane bilayers, this project aims to structurally characterize the assembly of ?-defensin 3, an important component of the innate immune system that helps fight microbial infections and may endogenously kill cancer cells with changes in membrane fluidity, with phospholipids using electron microscopy. This structure will provide a molecular mechanism by which defensin can disrupt membrane bilayers. Computational protein design will be used to critically evaluate our understanding of this mechanism. The design of de novo proteins that form highly-curved assemblies on membranes will test how this curvature contributes to the ability for these proteins to disrupt membranes. Tuning the assembly dynamics and membrane affinity will reveal how these thermodynamic parameters affect the function of membrane-deforming proteins. These designs would not only verify the molecular mechanism of defensins, but would be the first demonstration of the assembly of de novo designed proteins on membranes, forging new directions in computational protein design. In summary, this proposal addresses a fundamental question concerning the mechanism of membrane-disrupting proteins using electron microscopy and a computational protein design approach that probes the biophysical basis of this process.